Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors

Definitions

• the present disclosure is directed to neonatal Neutrophil Inhibitory Factor (nNIF) and nNIF-Related Peptides (NRPs).
• nNIF neonatal Neutrophil Inhibitory Factor
• NRPs nNIF-Related Peptides
• the present disclosure is also directed to methods of using nNIF and NRPs for the inhibition of neutrophil extracellular trap (NET) formation.
• nNIF and NRPs can be used for the treatment of and the prophylaxis against inflammatory disorders and cancer.
• NETs neutrophil extracellular traps
• PMNs polymorphonuclear leukocytes
• NET formation occurs by a novel cell death process often called NETosis, although "vital” NETosis, in which the neutrophils do not immediately die, has also been described (see Yipp BG, et al., Blood. 2013;122 (16):2784-94 and Yipp BG, et al., Nature medicine. 2012; 18 (9): 1386-93).
• the molecular mechanisms leading to NET formation have not been completely dissected and may depend in part on the stimulus (see Sorensen OE, et al., Journal of clinical investigation. 2016; 126 (5): 1612-20; Brinkmann V, et al., J Cell Biol. 2012;198 (5):773-83; Yipp BG, et al., Blood.
• NET-mediated capture and elimination of pathogens may complement traditional PMN antimicrobial activities including phagocytosis and intracellular killing (see Brinkmann V, et al., J Cell Biol. 2012; 198 (5):773-83 and Nauseef WM, Immunol Rev. 2007;219 (88-102)).
• Clinical observations indicate that defects in NET formation contribute to intractable infections in some instances (see Brinkmann V, et al., J Cell Biol. 2012;198 (5):773-83 and Bianchi M, et al., Blood.
• NET formation may be an important maladaptive activity of neutrophils (see Sorensen OE, et al., Journal of clinical investigation. 2016; 126 (5): 1612-20) if it is triggered inappropriately or is unregulated in infection and inflammation.
• LPS lipopolysaccharide
• FIG. 1 B is two images depicting neutrophils isolated from cord blood of a healthy term neonate on the day of delivery (left panel) or from venous blood on day 2 after birth (right panel) that were stimulated with LPS (100 ng/mL, 1 hour) and imaged as in FIG. 1A. Analysis of NET formation by neutrophils from a second term neonate yielded the same pattern.
• FIGS. 1A-1 D indicate that a NET-lnhibitory Factor is present in human umbilical cord blood.
• FIG. 2A is a provisional partial sequence of nNIF from mass spectroscopy, and published sequences of CRISPP (see Cercek L, et al. Cancer Detect Prey. 1992; 16 (5-6):305-19) and A1AT.
• FIG. 2D is an image depicting full-length A1AT, synthetic nNIF, and samples of depleted cord blood plasma and eluate studied in FIG. 2C that were subjected to western blotting using the carboxy-terminus A1 AT antibody.
• Full-length A1AT 52 kDa was not detected on this 16.5% Tris-tricine gel due to its size.
• control medium second panel
• A1AT full-length A1AT
• synthetic nNIF 1 nM
• NET-associated histone H3 content is depicted in the graph.
• FIGS. 2A-2E indicate that nNIF and related NRPs represent a family of NET-lnhibitory Peptides.
• FIG. 3A depicts LPS (100 ng/mL) activation. **P ⁇ 0.05 for LPS and CRISPP-SCR/LPS compared to control, ⁇ P ⁇ 0.05 for CRISPP/LPS and nNIF/LPS compared to both LPS and CRISPP-SCR/LPS.
• FIG. 3B depicts phorbol myristate acetate (PMA) (20 nM) activation. *P ⁇ 0.05 for both nNIF/PMA and CRISPP/PMA compared to PMA or CRISPP- SCR/PMA; **P ⁇ 0.01 for CRISPP/PMA versus CRISPP-SCR/PMA.
• PMA phorbol myristate acetate
• FIG. 3C depicts S. aureus (SA; MOI 100: 1 ) activation. *P ⁇ 0.05 for CRISPP/SA compared to SA or CRISPP-SCR/SA.
• FIG. 3D depicts dengue virus (MOI 0.05: 1 ) activation.
• Viral culture medium alone served as a "mock” control (left panels) for dengue virus.
• the PMNs were immediately fixed in the incubation medium (2% paraformaldehyde) prior to imaging, and quantitation of histone H3 release was not possible.
• FIG. 3E depicts Heme (1 ⁇ ). *P ⁇ 0.05 for Heme, LPS, and CRISPP- SCR/Heme versus control; ⁇ P ⁇ 0.05 for CRISPP/Heme versus Heme.
• FIGS. 3A-3E indicate that nNIF and the NRP CRISPP inhibit in vitro NET formation triggered by a spectrum of NET-inducing agonists. Neutrophils from venous blood of healthy adults were preincubated in medium alone or with nNIF, CRISPP, or CRISPP-SCR (all 1 nM) for 1 hour, and activated with the indicated agonists (n>3 for each), and NET formation was assessed after 1 hour of incubation as in FIG.
• NETs were also intact after treatment with nNIF or CRISPP but dismantled by DNase.
• FIGS. 4A and 4B indicate that NRPs do not dismantle NETs.
• FIG. 5B is a series of images depicting, in parallel, neutrophils that were preincubated with synthesized A1ATm 358 or scrambled A1 ATm 358 (A1 ATm 358 -SCR) (1 , 10, or 100 nM, 1 hour), activated with LPS, and NETs that were assessed by live cell imaging after incubation for 1 hour.
• a second experiment yielded a similar concentration-dependent pattern of inhibition by A1 ATm but not A1 ATm -SCR.
• FIGS. 5A and 5B indicate that A1 ATm 358 inhibits NET formation.
• FIG. 6A is a graph depicting results, after preincubation, of neutrophils that were stimulated with LPS (100 ng/mL) to trigger NET formation and incubated with a pathogenic isolate of E. coli. Total, phagocytic, and NET-mediated bacterial killing were measured (see Yost CC, et al. , Blood. 2009; 1 13 (25):6419-27).
• LPS 100 ng/mL
• NET-mediated bacterial killing were measured (see Yost CC, et al. , Blood. 2009; 1 13 (25):6419-27).
• FIG. 6B is a graph depicting reactive oxygen species generation that was measured by dihydrorhodamine detection after LPS stimulation (100 ng/mL, 1 hour).
• FIG. 6C is a graph depicting phagocytosis of fluorescently-labeled E. coli bioparticles that were measured by microscopy after a 4 hour incubation. Treatment with cytochalasin B and D served as a control for inhibition of phagocytosis. Student's t test, * P ⁇ 0.05.
• FIG. 6D is a graph depicting chemotaxis in response to IL-8 (2 ng/mL) that was examined in a Boyden chamber assay.
• the dashed line indicates the response to IL-8 alone arbitrarily set at 1.
• FIG. 6E is a graph depicting surface translocation of P-selectin on platelets activated by thrombin (0.1 U/mL) that was measured by flow cytometry. The dashed line indicates surface P-selectin on unstimulated platelets.
• FIG. 6F is a graph depicting formation of platelet-neutrophil aggregates that was measured after mixing of platelets activated with thrombin (0.1 U/mL) and neutrophils activated with LPS (100 ng/mL). The dashed line indicates control aggregate formation in response to LPS stimulation of the PMNs alone. * P ⁇ 0.05 for CRISPP and CRISPP-SCR compared to control.
• FIGS. 6A-6F indicate that NRPs selectively inhibit NET formation.
• FIG. 7C is a graph depicting nNIF (1 nM), nNIF-SCR (1 nM), and Cl- amidine (10 ⁇ ) that were examined in a cell-free deimination assay employing recombinant PAD4 and a synthetic substrate.
• nNIF 1 nM
• nNIF-SCR 1 nM
• Cl- amidine 10 ⁇
• IMAGEJTM software mean citrullinated-histone H3 pixel area per cell ⁇ SEM
• FIG. 7E is a series of images depicting neutrophils that were incubated with FLAG-tagged CRISPP-FLAG or CRISPP-SCR-FLAG (1 nM for both) for 1 hour, activated with LPS (100 ng/mL) for a further 2 hours, and then examined by confocal microscopy using an anti-FLAG antibody (n-3).
• the FLAG-tagged peptides were not internalized by isolated human platelets (unpublished experiments).
• FIGS. 7A-7E indicate that NRPs inhibit nuclear decondensation and histone citrullination in activated neutrophils.
• FIGS. 8A and 8B depict C57BL/6 mice that were not pretreated or pretreated with nNIF, CRISPP, or CRISPP-SCR (10 mg/kg i.p.; 1 hour) and were inoculated with E. coli (4.5 ⁇ 10 7 bacteria i.p.). After 3 hours, the animals were sacrificed, and peritoneal fluid (FIG. 8A) and membranes (FIG. 8B) were collected for analysis.
• FIG. 8C depicts C57BL/6 mice that were not pretreated (left two bars) or that were pretreated with CRISPP, nNIF, or CRISPP-SCR, and that were inoculated with E. coli i.p. as in FIGS. 8A and 8B.
• Neutrophil numbers in peritoneal fluid were counted after 3 hours (3-5 mice/group).
• One way ANOVA with Neuman-Keul's post hoc testing P ⁇ 0.05 for CRISPP versus CRISPP-SCR or not pretreated; * P ⁇ 0.05 for control versus all other groups.
• FIG. 8D depicts C57BL/6 mice that were pretreated with CRISPP or CRISPP-SCR and that were inoculated with E. coli as in FIGS. 8A and 8B (5 animals/group). After 3 hours, bacteria colony forming units (cfu) in the peritoneal fluid were measured (single-tailed Mann-Whitney test; * P ⁇ 0.05).
• FIG. 8E depicts peritoneal fluid NET formation, imaged and measured as in FIG. 8A (10 mice/group). *P ⁇ 0.05 for CRISPP/E. coli and nNIF/E. coli compared to CRISPP-SCR/E.co// ' and E.coli.
• FIG. 8F depicts NET formation on peritoneal membrane surfaces, imaged and quantitated as in FIG. 8B (3 mice in each group).
• *P ⁇ 0.05 for E.coli versus control red dashed line
• ⁇ P ⁇ 0.05 for CRISPP/E. coli and nNIF/E.co// ' versus CRISPP-SCR/E.co// ' One way ANOVA with Tukey's post-hoc testing applied in FIGS. 8E and 8F.
• FIGS. 8E and 8F One way ANOVA with Tukey's post-hoc testing applied in FIGS. 8E and 8F.
• FIGS. 8E and 8F Swiss-Webster mice that were not pretreated or that were pretreated with nNIF, CRISPP, or CRISPP-SCR were inoculated with E. coli i.p. as in FIGS. 8A and 8B. After 3 hours, peritoneal fluid and membranes were collected.
• FIGS. 8A-8F indicate that nNIF and CRISPP inhibit in vivo NET formation.
• FIG. 9A is a graph depicting C57BL/6 mice that were challenged with LPS (25 mg/kg i.p.).
• FIG. 9A is a graph depicting C57BL/6 mice that were challenged with LPS (25 mg/kg i.p.).
• CRISPP, nNIF, or CRISPP-SCR (10 mg/kg i.p.) was given 1 hour before and 6 hours after LPS. Animals with no treatments or given LPS alone were studied in parallel (n>10 mice for each condition).
• FIG. 9B is a graph depicting C57BL/6 mice that were subjected to cecal ligation and puncture (CLP).
• CLP cecal ligation and puncture
• FIG. 9C is a graph depicting mice that were assessed for severity of systemic illness in FIG. 9B and were then followed daily, where survivors were sacrificed at 144 hours. Log-rank (Mantel-Cox) statistical tool used. ** P ⁇ 0.01.
• FIGS. 9A-9C indicate that nNIF and CRISPP improve survival in experimental systemic inflammation.
• FIG. 10 is an image depicting that a low molecular weight peptide recognized by an antibody against the carboxy-terminus of A1AT is detected in umbilical cord blood samples but not plasma from adults. Samples of cord blood plasma from four healthy term neonates and venous blood samples from four healthy adult volunteers were examined by western blotting. As stated above, this is the full gel from which the left panel of FIG. 2B was prepared.
• FIG. 1 1 is a series of images depicting that nNIF and CRISPP inhibit NET formation at nanomolar concentrations.
• PMNs from healthy adult volunteers were preincubated in medium alone or with nNIF or CRISPP in the indicated concentrations for 1 hour.
• LPS 100 ng/mL
• red fluorescence NETs
• NE neutrophil elastase
• CRISPP or CRISPP-SCR both 1 nM
• PMA 20 nM
• Nuclear area was quantified using IMAGE JTM software (nuclear pixel area per cell ⁇ SEM).
• FIG. 12B depicts NE enzyme activity that was examined by cleavage of the synthetic substrate (MeOSuc)-AAPV-(pNA) to (MeOSuc)-AAPV and (pNA) as products detected by liquid chromatography and chromatogram peak identification by mass spectroscopy.
• (MeOSuc)-AAPV-(pNA) 160 pm was incubated with NE (2 mU), sivelestat (160 m), or NE and sivelestat (left panel) or with NE (2 mU), nNIF (10 nM), or NE and nNIF (right panel) for 3 hours at 37 °C. Chromatograms are offset on the X and Y axes for ease of comparison. In the presence of NE alone, the (MeOSvc)-AAPV-(pNA) peak was almost completely eliminated and (MeOSvc)- AAPV and (pNA) peaks were generated. In the presence of sivelestat, this substrate cleavage was inhibited but not eliminated.
• FIGS. 12A and 12B indicate that NE mediates nuclear decondensation but nNIF and CRISPP do not inhibit NE activity in vitro.
• FIG. 13 is a series of images depicting that FLAG-tagged CRISPP inhibits NET formation by LPS-activated neutrophils.
• FIG. 14 depicts that a protease, high temperature requirement A1 (HTRA1 ), is upregulated in the human placenta in the third trimester of pregnancy and cleaves A1AT in the C-terminus, generating a fragment somewhat larger in size but including the sequence of nNIF (see Frochaux V, et a/., Plos one. 9(10): e109483. doi:10.1371 ).
• the peptide generated by cleavage of A1AT by HTRA1 was synthesized and it was found that the peptide inhibits NET formation.
• This disclosure relates to neonatal Neutrophil Inhibitory Factor (nNIF) and nNIF-Related Peptides (NRPs).
• the disclosure is also related to methods of using nNIF and NRPs for the inhibition of neutrophil extracellular trap (NET) formation.
• nNIF and NRPs can be used for the treatment of and the prophylaxis against inflammatory disorders and cancer.
• a "NET-lnhibitory Peptide (NIP)” is an anti-inflammatory agent that inhibits neutrophil extracellular trap (NET) formation.
• NIPs include, but are not limited to: neonatal NET-lnhibitory Factor (nNIF); a pharmaceutically acceptable salt of nNIF; an analog of a naturally occurring form of nNIF, which nNIF analog inhibits NETosis and/or the formation of NETs and is structurally altered, relative to a given human nNIF, by at least one amino acid addition, deletion, or substitution, or by incorporation of one or more amino acids with a blocking group; a pharmaceutically acceptable salt of a nNIF analog; a nNIF-Related Peptide (NRP); a pharmaceutically acceptable salt of a NRP; a NRP analog; or a pharmaceutically acceptable salt of a NRP analog.
• a "neonatal Neutrophil Inhibitory Factor peptide” or “nNIF peptide” is defined herein as a nNIF which is naturally occurring in mammals.
• a "neonatal NIF-Related Peptide” or “NRP” is defined herein as a Cancer- Associated SCM-Recognition, Immune Defense Suppression, and Serine Protease Protection Peptide (CRISPP) which is naturally occurring in humans; A1ATm 358 , which has been shown to inhibit NET formation; HTRA1 -CF; other nNIF-Related Peptides; and analogs of naturally occurring forms of NRPs that inhibit NETosis and/or the formation of NETs and are structurally altered, relative to a given human NRP, by at least one amino acid addition, deletion, or substitution, or by incorporation of one or more amino acids with a blocking group.
• CRISPP Serine Protease Protection Peptide
• Inflammatory disorders are defined herein as disorders characterized by pathological inflammation. Inflammatory disorders include, but are not limited to, conditions associated with infection, autoimmunity, and allergy. Inflammatory disorders as defined herein may include, but are not limited to, acute respiratory distress syndrome (ARDS); bronchopulmonary dysplasia (BPD); chronic obstructive pulmonary disease (COPD); cystic fibrosis; inflammation in cancer and its complications; inflammatory bowel disease (IBD); inflammatory lung disease (ILD); influenza-induced pneumonitis; necrotizing enterocolitis (NEC); neonatal chronic lung disease (CLD); periodontitis; pre-eclampsia; retinopathy of prematurity (ROP); sepsis; systemic inflammatory response syndrome (SIRS); thrombosis; transfusion- related acute lung injury (TRALI); vasculitis; autoimmune syndromes including, but not limited to, rheumatoid arthritis (RA), systemic lupus ery
• PMN polymorphonuclear leukocyte
• NIPs NET Inhibitory Peptides
• nNIF neonatal NET-lnhibitory Factor
• NRPs nNIF-Related Peptides
• the NIPs, nNIF, nNIF analogs, NRPs, and/or NRP analogs may be used for inhibiting NETosis and/or the formation of neutrophil extracellular traps (NETs).
• NETs neutrophil extracellular traps
• a first aspect of the disclosure relates to methods of treating inflammatory disorders.
• this disclosure provides methods of treating a patient having an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NIP, or a pharmaceutically acceptable salt of a N IP, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the inflammatory disorder.
• the pathological effects or symptoms may include one or more of the following: pain, heat, redness, swelling and/or edema, hypotension, fibrosis and/or postinflammatory fibrosis, end organ failure (i.e. , renal, cardiac, hepatic), tissue damage, and/or loss of function.
• this disclosure provides methods of treating a patient having an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF, or a pharmaceutically acceptable salt of a nNIF, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides methods of treating a patient having an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF analog, or a pharmaceutically acceptable salt of a nNIF analog, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides methods of treating a patient having an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP, or a pharmaceutically acceptable salt of a NRP, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides methods of treating a patient having an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP analog, or a pharmaceutically acceptable salt of a NRP analog, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the inflammatory disorder.
• the patient may be a mammal. In certain embodiments the patient may be a human. Any patient or subject requiring inhibition of NETosis and/or NET formation may potentially be a candidate for treatment with a NIP, a pharmaceutically acceptable salt of a NIP, a nNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a NRP, a pharmaceutically acceptable salt of a NRP, a NRP analog, and/or a pharmaceutically acceptable salt of a NRP analog.
• the inflammatory disorder may at least partially involve or be at least partially caused by neutrophil extracellular trap (NET) formation and/or NETosis.
• NET neutrophil extracellular trap
• the inflammatory disorder may be an acute inflammatory disorder, a chronic inflammatory disorder, and/or an immune disorder.
• the inflammatory disorder may be an autoimmunity disorder.
• the inflammatory disorder may be a disorder of coagulation.
• the inflammatory disorder may be one or more of, but is not limited to, acute respiratory distress syndrome (ARDS); bronchopulmonary dysplasia (BPD); chronic obstructive pulmonary disease (COPD); cystic fibrosis; inflammation in cancer and its complications; inflammatory bowel disease (IBD); inflammatory lung disease (ILD); influenza-induced pneumonitis; necrotizing enterocolitis (NEC); neonatal chronic lung disease (CLD); periodontitis; preeclampsia; retinopathy of prematurity (ROP); sepsis; systemic inflammatory response syndrome (SIRS); thrombosis; transfusion-related acute lung injury (TRALI); vasculitis; autoimmune syndromes including, but not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Wegener's granulomatosis (WG); and disorders of nonresolved inflammation.
• ARDS acute respiratory distress syndrome
• BPD broncho
• this disclosure relates to methods for treating a patient having a cancer.
• the cancer may be at least one of melanoma, ovarian cancer, stomach cancer, lung cancer, or another suitable cancer.
• the methods for treating a patient having a cancer may include administering to the patient an effective amount of a pharmaceutical composition including a NIP.
• the pharmaceutical composition may also include a NIP and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may reduce, or be configured to reduce, a pathological effect or symptom of the cancer.
• the NIP may be one of a nNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a NRP, a pharmaceutically acceptable salt of a NRP, a NRP analog, and/or a pharmaceutically acceptable salt of a NRP analog.
• the pharmaceutical composition may inhibit or substantially inhibit NET formation.
• the pharmaceutical composition may, at least in part, reduce a pathological effect or symptom of the cancer by inhibiting NET formation in the patient having cancer.
• the patient may be a mammal, such as a human.
• this disclosure relates to methods for treating a patient at risk of developing a cancer.
• the cancer may be at least one of melanoma, ovarian cancer, stomach cancer, lung cancer, or another suitable cancer.
• the methods for treating a patient at risk of developing a cancer may include administering to the patient an effective amount of a pharmaceutical composition including a NIP.
• the pharmaceutical composition may also include a NIP and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may reduce, or be configured to reduce, the risk of developing the cancer.
• the NIP may be one of a nNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a NRP, a pharmaceutically acceptable salt of a NRP, a NRP analog, and/or a pharmaceutically acceptable salt of a NRP analog.
• the pharmaceutical composition may inhibit or substantially inhibit NET formation.
• the pharmaceutical composition may, at least in part, reduce the risk of developing the cancer by inhibiting NET formation in the patient at risk of developing the cancer.
• the patient may be a mammal, such as a human.
• this disclosure relates to methods for inhibiting, or substantially inhibiting, metastasis in a patient having cancer.
• the cancer may be at least one of melanoma, ovarian cancer, stomach cancer, lung cancer, or another suitable cancer.
• the methods for inhibiting metastasis may include administering to the patient an effective amount of a pharmaceutical composition including a NIP.
• the pharmaceutical composition may also include a NIP and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may reduce, or be configured to reduce, the risk of metastasis in the patient.
• the NIP may be one of a nNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a NRP, a pharmaceutically acceptable salt of a NRP, a NRP analog, and/or a pharmaceutically acceptable salt of a NRP analog.
• the pharmaceutical composition may inhibit or substantially inhibit NET formation.
• the pharmaceutical composition may, at least in part, reduce the risk of metastasis by inhibiting NET formation in the patient having a cancer.
• the patient may be a mammal, such as a human.
• the pharmaceutical composition comprising the NIP may not globally depress functions of PMNs.
• the functions of PMNs include, but are not limited to, chemotaxis, phagocytosis, reactive oxygen species (ROS) generation, cytokine/chemokine synthesis and secretion, NET formation/NETosis, and/or intracellular/extracellular bacterial killing.
• the pharmaceutical composition may not inhibit or substantially inhibit PMN phagocytosis.
• the pharmaceutical composition may not inhibit or substantially inhibit PMN chemotaxis.
• the pharmaceutical composition may not inhibit or substantially inhibit generation of ROS.
• the pharmaceutical composition may not inhibit or substantially inhibit PMN intracellular bacterial killing. Accordingly, administration of the pharmaceutical composition comprising the NIP to treat a patient having cancer, or at risk of developing cancer, may avoid some of the side effects of chemotherapy regimens used in the treatment of or prophylaxis against cancer.
• this disclosure relates to methods for treating patients having a cancer, wherein the methods include identifying NET formation at or adjacent cancerous cells in a patient.
• the methods may further include administering to the patient having NET formation (e.g., the patient wherein NET formation is identified) an effective amount of a pharmaceutical composition including a NIP and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the cancer.
• the methods may include obtaining a sample of cancerous cells from the patient. The cancerous cells, or the sample including cancerous cells, can be examined and/or tested to identify the presence of NET formation.
• this disclosure relates to methods for treating patients at risk of developing cancer.
• the methods may include identifying NET formation in a patient.
• the methods may include administering to the patient an effective amount of a pharmaceutical composition including a NIP and a pharmaceutically acceptable carrier, to reduce the patient's risk of developing the cancer.
• the pharmaceutical composition may substantially inhibit NET formation and/or NETosis. In other embodiments, the pharmaceutical composition may inhibit or substantially inhibit NET-mediated inflammatory tissue damage.
• this disclosure relates to methods of diagnosing a patient having cancer who would benefit from treatment with a NET-lnhibitory Peptide (NIP).
• the method may include obtaining a sample of cancerous cells from the patient, detecting whether NET formation is present in the sample of cancerous cells, and/or diagnosing the patient as a patient who would benefit from treatment with a NIP when the presence of NET formation in the sample of cancerous cells is detected.
• the cancerous cells may include at least one of melanocytes, ovarian cells, stomach cells, lung cells, or another suitable type of cancerous cells.
• this disclosure relates to methods of diagnosing and treating a patient having cancer who would benefit from treatment with a NET- lnhibitory Peptide (NIP).
• the method may include obtaining a sample of cancerous cells from the patient, detecting whether NET formation is present in the sample of cancerous cells, diagnosing the patient as a patient who would benefit from treatment with a NIP when the presence of NET formation in the sample of cancerous cells is detected, and/or administering an effective amount of a pharmaceutical composition comprising a NIP to the diagnosed patient.
• the NIP may be one of neonatal NET-lnhibitory Factor (nNIF), a pharmaceutically acceptable salt of nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a nNIF-Related Peptide (NRP), a pharmaceutically acceptable salt of a NRP, a NRP analog, and/or a pharmaceutically acceptable salt of a NRP analog.
• the cancerous cells may include at least one of melanocytes, ovarian cells, stomach cells, lung cells, or another suitable type of cancerous cells.
• the pharmaceutical composition may substantially inhibit NET formation.
• the patient may be a mammal. In some embodiments, the patient may be a human.
• NIP nNIF
• nNIF analog nNIF analog
• NRP NRP analog
• salt thereof selected for inhibiting NETosis and/or NET formation can be prepared by a variety of techniques known for generating peptide products.
• vertebrate forms of nNIF and NRP can be obtained by extraction from the natural source, using an appropriate combination of protein isolation techniques. Other techniques are also within the scope of this disclosure.
• NIPs, nNIF, nNIF analogs, NRPs, NRP analogs, and/or salts thereof can be synthesized using standard techniques of peptide chemistry and can be assessed for inhibition of NETosis and/or NET formation activity.
• the selected NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof can be prepared by a variety of techniques for generating peptide products.
• Those NIPs, nNIF, nNIF analogs, NRPs, NRP analogs, and/or salts thereof that incorporate only L-amino acids can be produced in commercial quantities by application of recombinant DNA technology.
• DNA coding for the desired NIP, nNIF, nNIF analog, NRP, and/or NRP analog is incorporated into an expression vector and transformed into a host cell (e.g., yeast, bacteria, or a mammalian cell), which is then cultured under conditions appropriate for NIP, nNIF, nNIF analog, NRP, and/or NRP analog expression.
• a host cell e.g., yeast, bacteria, or a mammalian cell
• a variety of gene expression systems have been adapted for this purpose, and typically drive expression of the desired gene from expression regulatory elements used naturally by the chosen host.
• NIP 9-fluoroenylmethyloxycarbonyl
• f-Boc tert-butyloxycarbonyl
• the peptide may then be purified to ensure the recovery of a single oligopeptide having the selected amino acid sequence. Purification may be achieved using any of the standard approaches, which include, but are not limited to, reversed-phase high-pressure liquid chromatography (RP-HPLC) on alkylated silica columns (e.g., C4-, C8-, or C18-silica). Such column fractionation is generally accomplished by running linear gradients (e.g., 10-90%) of increasing percentage organic solvent (e.g.
• aqueous buffer usually containing a small amount (e.g., 0.1 %) of pairing agent such as trifluoroacetic acid (TFA) or triethanolamine (TEA).
• pairing agent such as trifluoroacetic acid (TFA) or triethanolamine (TEA).
• ion- exchange HPLC can be employed to separate peptide species on the basis of their charge characteristics. Column fractions are collected, and those containing peptide of the desired and/or required purity are optionally pooled.
• the NIP, nNIF, nNIF analog, NRP, and/or NRP analog may then be treated in the established manner to exchange the cleavage acid (e.g. , TFA) with a pharmaceutically acceptable acid, such as acetic, hydrochloric, phosphoric, maleic, tartaric, succinic, and the like, to generate a pharmaceutically acceptable acid addition salt of the peptide.
• a pharmaceutically acceptable acid such as acetic, hydrochloric, phosphoric,
• Analogs of human NIPs, nNIF, and/or NRPs can be generated using standard techniques of peptide chemistry and can be assessed for inhibition of NETosis and/or NET formation activity, all according to the guidance provided herein.
• the analogs are based, at least in part, upon the sequences of human nNIF (SEQ ID NO:1 ), CRISPP (SEQ ID NO:2), A1 ATm 358 (SEQ ID NO:3), and/or HTRA1 -CF (SEQ ID NO:4) as follows (wherein X can be any naturally occurring amino acid):
• KFNKPFVFLMIEQNTKSPLFMGKWNPTQ (SEQ ID NO: 1 )
• any substitution, addition, or deletion of an amino acid or amino acids of a NIP, nNIF, and/or NRP that does not destroy the NET-inhibitory activity of the NIP, nNIF, and/or NRP may be usefully employed in this disclosure.
• the NIP, nNIF, and/or NRP analogs are at least as active as the native human NIP, nNIF, and/or NRP.
• NET-inhibitory activity may be determined in vitro as described in this disclosure.
• the NIP, nN IF, and/or NRP analog has one or more enhanced properties compared with the native human NIP, nNIF, and/or NRP.
• such analogs may exhibit enhanced serum stability, enhanced receptor binding, and enhanced signal-transducing activity.
• Other modifications to NIPs, nNIF, nNIF analogs, NRPs, and/or NRP analogs that may usefully be employed in this disclosure are those which render the molecule resistant to oxidation.
• a researcher may determine whether a particular NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof may be used to treat an inflammatory disorder by administering the peptide or analog to individuals who have the inflammatory disorder. The researcher may then determine, using diagnostic biomarkers, whether the individuals thus treated show decreased inflammation and improvement of the inflammatory condition.
• the disclosure also encompasses non-conservative substitutions of amino acids in any vertebrate NIP, nNIF, and/or NRP sequence, provided that the non-conservative substitutions occur at amino acid positions known to vary in NIPs, nNIF, and/or NRPs isolated from different species.
• Non-conserved residue positions are readily determined by aligning known vertebrate NIP, nNIF, and/or NRP sequences.
• the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof may be provided in pharmaceutically acceptable form (e.g., as a preparation that is sterile-filtered, e.g. , through a 0.22 ⁇ filter, and substantially pyrogen-free). It may be desired that the NIP, nNIF, and/or NRP peptide to be formulated migrates as a single or individualized peak on HPLC, exhibits uniform and authentic amino acid composition and sequence upon analysis thereof, and otherwise meets standards set by the various national bodies which regulate quality of pharmaceutical products.
• pharmaceutically acceptable form e.g., as a preparation that is sterile-filtered, e.g. , through a 0.22 ⁇ filter, and substantially pyrogen-free. It may be desired that the NIP, nNIF, and/or NRP peptide to be formulated migrates as a single or individualized peak on HPLC, exhibits uniform and authentic amino acid composition and sequence upon analysis thereof, and otherwise meets standards set by
• the aqueous carrier or vehicle may be supplemented for use as an injectable with an amount of gelatin that serves to depot the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof at or near the site of injection, for its slow release to the desired site of action. Concentrations of gelatin effective to achieve the depot effect are expected to lie in the range from 10% to 20%. Alternative gelling agents, such as hyaluronic acid (HA), may also be useful as depoting agents.
• HA hyaluronic acid
• the NIPs, nNIF, nNIF analogs, NRPs, NRP analogs, and/or salts thereof of the present disclosure may also be formulated as slow-release implantation formulations for extended and sustained administration of the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof.
• sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
• the structure, selection, and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including Domb et ai, Polym Advan Technol, 1992, 3: 279-292.
• Liposomes may also be used to provide for the sustained release of a nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. Nos.
• Sustained release formulations may be of particular interest when it is desirable to provide a high local concentration of a NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof (e.g., near the site of inflammation to inhibit NETosis and/or NET formation, etc.).
• NIPs, nNIF, nNIF analogs, NRPs, NRP analogs, and/or salts thereof of the present disclosure may also be incorporated into a device or devices, both implanted or topical, for extended and sustained administration of the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof.
• the chosen NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof may be formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivering the peptide by the chosen route of administration.
• Suitable pharmaceutically acceptable carriers are those used conventionally with peptide-based drugs, such as diluents, excipients and the like. Reference may be made to Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London, UK.
• the compounds may be formulated for administration by infusion or by injection (e.g.
• the compounds may be administered in a vehicle such as distilled water, saline, phosphate buffered saline, or 5% dextrose solution.
• a solubility enhancer such as acetic acid.
• Another aspect of the disclosure relates to methods of treating complications of prematurity.
• this disclosure provides for methods of treating a patient having a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NIP, or a pharmaceutically acceptable salt of a NIP, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the complication of prematurity, such as the prolonged need for oxygen support associated with neonatal chronic lung disease or the need for surgical intervention or prolonged total parenteral nutrition in infants that develop necrotizing enterocolitis.
• this disclosure provides for methods of treating a patient having a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF, or a pharmaceutically acceptable salt of a nNIF, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the complication of prematurity.
• the disclosure provides methods of treating a patient having a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF analog, or a pharmaceutically acceptable salt of a nNIF analog, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the complication of prematurity.
• the disclosure provides methods of treating a patient having a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP, or a pharmaceutically acceptable salt of a NRP, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the complication of prematurity.
• the disclosure provides methods of treating a patient having a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP analog, or a pharmaceutically acceptable salt of a NRP analog, and a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of the complication of prematurity.
• the patient may be a mammal. In certain embodiments, the patient may be a human.
• the complication of prematurity may at least partially involve or be at least partially caused by neutrophil extracellular trap (NET) formation and/or NETosis.
• the pharmaceutical composition may substantially inhibit NET formation and/or NETosis.
• the pharmaceutical composition may inhibit or substantially inhibit NET-mediated inflammatory tissue damage.
• the complication of prematurity may be one or more of, but not limited to, necrotizing enterocolitis (NEC), respiratory distress syndrome (RDS), pneumonia, bronchopulmonary dysplasia (BPD), neonatal chronic lung disease (CLD), neurodevelopmental delay, retinopathy of prematurity (ROP), and/or sepsis.
• NEC necrotizing enterocolitis
• RDS respiratory distress syndrome
• BPD bronchopulmonary dysplasia
• CLD neonatal chronic lung disease
• ROP retinopathy of prematurity
• sepsis sepsis
• a further aspect of the disclosure relates to methods of prophylaxis against inflammatory disorders.
• the disclosure provides for methods of prophylaxis against an inflammatory disorder in a patient at risk of developing an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NIP, or a pharmaceutically acceptable salt of a NIP, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides for methods of prophylaxis against an inflammatory disorder in a patient at risk of developing an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF, or a pharmaceutically acceptable salt of a nNIF, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides for methods of prophylaxis against an inflammatory disorder in a patient at risk of developing an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF analog, or a pharmaceutically acceptable salt of a nNIF analog, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides for methods of prophylaxis against an inflammatory disorder in a patient at risk of developing an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP, or a pharmaceutically acceptable salt of a NRP, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the inflammatory disorder.
• the disclosure provides for methods of prophylaxis against an inflammatory disorder in a patient at risk of developing an inflammatory disorder, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP analog, or a pharmaceutically acceptable salt of the NRP analog, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the inflammatory disorder.
• the patient may be a mammal, including a human.
• the inflammatory disorder may at least partially involve or be at least partially caused by neutrophil extracellular trap (NET) formation and/or NETosis.
• NET neutrophil extracellular trap
• the inflammatory disorder may be an acute inflammatory disorder.
• the inflammatory disorder may be a chronic inflammatory disorder.
• the inflammatory disorder may be an autoimmunity disorder.
• the inflammatory disorder may be a disorder of coagulation.
• the inflammatory disorder may be one or more of, but not limited to, the inflammatory disorders defined and/or listed above.
• the pharmaceutical composition may substantially inhibit NET formation and/or NETosis. In other embodiments, the pharmaceutical composition may inhibit or substantially inhibit NET-mediated inflammatory tissue damage.
• Another aspect of the disclosure relates to methods of prophylaxis against complications of prematurity.
• this disclosure provides methods of prophylaxis against complications of prematurity in a patient at risk of developing a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a neonatal NIP, or a pharmaceutically acceptable salt of a NIP, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the complication of prematurity.
• this disclosure provides methods of prophylaxis against complications of prematurity in a patient at risk of developing a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a neonatal nNIF, or a pharmaceutically acceptable salt of a nNIF, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the complication of prematurity.
• the disclosure provides methods of prophylaxis against complications of prematurity in a patient at risk of developing a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nN IF analog, or a pharmaceutically acceptable salt of a nNIF analog, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the complication of prematurity.
• the disclosure provides methods of prophylaxis against complications of prematurity in a patient at risk of developing a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a nNIF-Related Peptide (NRP), or a pharmaceutically acceptable salt of a NRP, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the complication of prematurity.
• NRP nNIF-Related Peptide
• the disclosure provides methods of prophylaxis against complications of prematurity in a patient at risk of developing a complication of prematurity, the methods comprising administering to the patient an effective amount of a pharmaceutical composition comprising a NRP analog, or a pharmaceutically acceptable salt of a NRP analog, and a pharmaceutically acceptable carrier to reduce the risk of developing a pathological effect or symptom of the complication of prematurity.
• the patient may be a mammal, including a human.
• the complication of prematurity may at least partially involve or be at least partially caused by neutrophil extracellular trap (NET) formation and/or NETosis.
• the pharmaceutical composition may substantially inhibit NET formation and/or NETosis.
• the pharmaceutical composition may inhibit or substantially inhibit NET-mediated inflammatory tissue damage.
• the complication of prematurity may be one or more of, but not limited to, necrotizing enterocolitis (NEC), respiratory distress syndrome (RDS), pneumonia, bronchopulmonary dysplasia (BPD), neonatal chronic lung disease (CLD), neurodevelopmental delay, retinopathy of prematurity (ROP), and/or sepsis.
• NEC necrotizing enterocolitis
• RDS respiratory distress syndrome
• BPD bronchopulmonary dysplasia
• CLD neonatal chronic lung disease
• ROP retinopathy of prematurity
• sepsis sepsis
• compositions comprising NIPs.
• the pharmaceutical composition may comprise neonatal NET-lnhibitory Factor (nNIF), or a pharmaceutically acceptable salt of a nNIF, and a pharmaceutically acceptable carrier.
• nNIF neonatal NET-lnhibitory Factor
• the pharmaceutical composition may comprise a nNIF analog, or a pharmaceutically acceptable salt of a nNIF analog, and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may comprise a nNIF-Related Peptide (NRP), or a pharmaceutically acceptable salt of a NRP, and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may comprise a NRP analog, or a pharmaceutically acceptable salt of a NRP analog, and a pharmaceutically acceptable carrier.
• the pharmaceutical composition may comprise nNIF (e.g. , human nNIF), or a salt thereof, and the nNIF, or the salt thereof, may comprise at least a portion of the amino acid sequence:
• KFNKPFVFLMIEQNTKSPLFMGKVVNPTQ (SEQ ID NO: 1 )
• the pharmaceutical composition may comprise CRISPP, or a salt thereof, and the CRISPP, or the salt thereof, may comprise at least a portion of the amino acid sequence:
• the pharmaceutical composition may comprise A1ATm 358 , or a salt thereof, and the A1ATm 358 , or the salt thereof, may comprise at least a portion of the amino acid sequence:
• the pharmaceutical composition may comprise HTRA1 -CF, or a salt thereof, and the HTRA1 -CF, or the salt thereof, may comprise at least a portion of the amino acid sequence:
• SIPPEVKFNKPFVFLMIEQNTKSPLFMGKWNPTQK SEQ ID NO:4
• At least one amino acid of the nNIF, the salt of the nNIF, the nNIF analog, the salt of the nNIF analog, the NRP, the salt of the NRP, the NRP analog, the salt of the NRP analog, the CRISPP, the salt of the CRISPP, the A1ATm 358 , the salt of the A1 ATm 358 , the HTRA1 -CF, or the salt of the HTRA1 -CF may be bound to a chemical modifier.
• the chemical modifier may be selected from at least one of a lipid, a polyethylene glycol (PEG), a saccharide, or any other suitable molecule.
• PEG polyethylene glycol
• Other chemical modifications of the pharmaceutical composition— for example, cationization, methylization, and cyclization— are also within the scope of this disclosure.
• Attachment of a lipid to the peptide may increase lipophilicity of the pharmaceutical composition.
• Attachment of a PEG to the peptide increases the molecular weight of the peptide.
• PEGylation may improve solubility of the pharmaceutical composition.
• PEGylation may reduce dosage frequency and/or toxicity of the pharmaceutical composition.
• PEGylation may extend circulating life of the pharmaceutical composition, and/or extend stability of the pharmaceutical composition, and/or may enhance protection of the pharmaceutical composition from proteolytic degradation. PEGylation may also reduce immunogenicity and/or antigenicity of the pharmaceutical composition.
• Attachment of one or more saccharides to the peptide may serve a variety of functional and/or structural roles in the pharmaceutical composition. Glycosylation may improve delivery of the pharmaceutical composition to a target or to targets of choice. Glycosylation may also reduce the toxicity of the pharmaceutical composition.
• the pharmaceutical composition comprising the nNIF, the salt of the nNIF, the nNIF analog, the salt of the nNIF analog, the NRP, the salt of the NRP, the NRP analog, or the salt of the NRP analog may be present in an amount effective to inhibit, or to substantially inhibit, damage selected from at least one of inflammatory tissue injury and/or inflammatory vascular injury.
• the pharmaceutical composition comprising the nNIF, the salt of the nNIF, the nNIF analog, the salt of the nNIF analog, the NRP, the salt of the NRP, the NRP analog, or the salt of the NRP analog may not globally depress functions of PMNs.
• the functions of PMNs include, but are not limited to, chemotaxis, phagocytosis, reactive oxygen species (ROS) generation, cytokine/chemokine synthesis and secretion, NET formation/NETosis, and/or intracellular/extracellular bacterial killing.
• the pharmaceutical composition may not inhibit or substantially inhibit PMN phagocytosis.
• the pharmaceutical composition may not inhibit or substantially inhibit PMN chemotaxis. In yet other embodiments, the pharmaceutical composition may not inhibit or substantially inhibit generation of ROS. In other embodiments, the pharmaceutical composition may not inhibit or substantially inhibit PMN intracellular bacterial killing.
• the pharmaceutical composition may comprise a nNIF analog, a salt of a nNIF analog, a NRP analog, or a salt of a NRP analog, and the analog or the salt of the analog may not be a naturally occurring analog or salt of the analog.
• the pharmaceutical composition may be present in an amount effective to inhibit, or to substantially inhibit, NET formation and/or NETosis.
• the NET formation and/or NETosis may be stimulated by a bacterium, a fungus, a parasite, a virus, and/or any other appropriate stimulator of NET formation and/or NETosis.
• the virus may be a hemorrhagic fever virus.
• Hemorrhagic fever viruses are described, e.g., in Borio et a/., JAMA, 2002, 287(18): 2391 -2405, and include, but are not limited to, filoviruses such as Ebola virus and Marburg virus, arenaviruses such as Lassa virus, hantaviruses, and flaviviruses such as dengue virus and yellow fever virus.
• the NET formation and/or NETosis may be stimulated by one or more bacterial species, including, but not limited to, Bacillus species, Escherichia species, Francisella species, Streptococcus species, Staphylococcus species, Yersinia species, and/or any other appropriate gram-negative or gram-positive bacterium or bacteria.
• Bacillus species may be Bacillus anthracis (anthrax).
• the Escherichia species may be Escherichia coli.
• the Francisella species may be Francisella tularensis (tularemia).
• the Staphylococcus species may be Staphylococcus aureus.
• the NET formation and/or NETosis may be stimulated by beta-defensin 1 , HIV-1 , lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and/or Staphylococcus aureus alpha-toxin.
• beta-defensin 1 HIV-1
• lipopolysaccharide LPS
• PMA phorbol myristate acetate
• Staphylococcus aureus alpha-toxin Staphylococcus aureus alpha-toxin.
• the pharmaceutical composition may comprise a NRP and/or a NRP analog.
• the pharmaceutical composition may comprise Cancer-Associated SCM-Recognition, Immune Defense Suppression, and Serine Protease Protection Peptide (CRISPP) and/or a CRISPP analog.
• CRISPP Serine Protease Protection Peptide
• the pharmaceutical composition may comprise A1ATm 358 and/or an A1ATm 358 analog.
• the pharmaceutical composition may comprise HTRA1 -CF and/or a HTRA1 -CF analog.
• the pharmaceutical may comprise another NRP.
• the NRP may be an isolated and purified component of umbilical cord blood.
• compositions for inhibiting the formation of NETs and/or NETosis in a mammal may comprise a nNIF, a pharmaceutically acceptable salt of the nNIF, a nNIF analog, a pharmaceutically acceptable salt of the nNIF analog, a NRP, a pharmaceutically acceptable salt of the NRP, a NRP analog, or a pharmaceutically acceptable salt of the NRP analog, and a pharmaceutically acceptable carrier.
• the mammal may be a human.
• this disclosure relates to a NET-lnhibitory Peptide (NIP).
• NIP NET-lnhibitory Peptide
• the NIP may be an isolated and purified nNIF protein comprising SEQ ID NO: 1 .
• the isolated and purified nNIF protein may comprise at least 24 contiguous amino acids of SEQ ID NO: 1 .
• the isolated and purified nNIF protein may comprise at least 12 contiguous amino acids of SEQ ID NO: 1 .
• the isolated and purified nNIF protein may comprise at least six contiguous amino acids of SEQ ID NO: 1 .
• the NIP may be an isolated and purified nNIF protein wherein the sequence may be at least 80% identical to SEQ ID NO: 1 .
• the isolated and purified nNIF may be at least 60% identical to SEQ ID NO:1 .
• the isolated and purified nNIF may be at least 40% identical to SEQ ID NO: 1 .
• the isolated and purified nNIF may be at least 20% identical to SEQ ID NO: 1 .
• the NIP may be an isolated and purified CRISPP protein comprising SEQ ID NO:2.
• the isolated and purified CRISPP protein may comprise at least 24 contiguous amino acids of SEQ ID NO:2.
• the isolated and purified CRISPP protein may comprise at least 12 contiguous amino acids of SEQ ID NO:2.
• the isolated and purified CRISPP protein may comprise at least six contiguous amino acids of SEQ ID NO:2.
• the NIP may be an isolated and purified CRISPP protein wherein the sequence may be at least 80% identical to SEQ ID NO:2. In other embodiments, the isolated and purified CRISPP may be at least 60% identical to SEQ ID NO:2. In yet other embodiments, the isolated and purified CRISPP may be at least 40% identical to SEQ ID NO:2. In still other embodiments, the isolated and purified CRISPP may be at least 20% identical to SEQ ID NO:2. [0145] In some embodiments, the NIP may be an isolated and purified A1 ATm protein comprising SEQ ID NO:3. In certain other embodiments, the isolated and purified A1ATm 358 protein may comprise at least 24 contiguous amino acids of SEQ ID NO:3.
• the isolated and purified A1ATm 358 protein may comprise at least 12 contiguous amino acids of SEQ ID NO:3. In still other embodiments, the isolated and purified A1ATm 358 protein may comprise at least six contiguous amino acids of SEQ ID NO:3.
• the NIP may be an isolated and purified A1ATm 358 protein wherein the sequence may be at least 80% identical to SEQ ID NO:3.
• the isolated and purified A1 ATm 358 may be at least 60% identical to SEQ ID NO:3.
• the isolated and purified A1ATm 358 may be at least 40% identical to SEQ ID NO:3.
• the isolated and purified A1ATm 358 may be at least 20% identical to SEQ ID NO:3.
• the NIP may be an isolated and purified HTRA1 - CF protein comprising SEQ ID NO:4.
• the isolated and purified HTRA1 -CF protein may comprise at least 24 contiguous amino acids of SEQ ID NO:4.
• the isolated and purified HTRA1 -CF protein may comprise at least 12 contiguous amino acids of SEQ ID NO:4.
• the isolated and purified HTRA1 -CF protein may comprise at least six contiguous amino acids of SEQ ID NO:4.
• the N IP may be an isolated and purified HTRA1 - CF protein wherein the sequence may be at least 80% identical to SEQ ID NO:4.
• the isolated and purified HTRA1 -CF may be at least 60% identical to SEQ ID NO:4.
• the isolated and purified HTRA1 -CF may be at least 40% identical to SEQ ID NO:4.
• the isolated and purified HTRA1 -CF may be at least 20% identical to SEQ ID NO:4.
• this disclosure relates to a nNIF protein analog, a CRISPP protein analog, an A1ATm 358 analog, and/or a HTRA1 -CF analog.
• the nNIF protein analog may be an isolated and purified nNIF analog
• the CRISPP protein analog may be an isolated and purified CRISPP analog
• the A1ATm 358 protein analog may be an isolated and purified A1 ATm 358 analog
• the HTRA1 -CF protein analog may be an isolated and purified HTRA1 -CF analog.
• An effective dosage and treatment protocol may be determined by conventional means, e.g., by starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Primary among these is whether any NIPs are normally circulating in the plasma and, if so, the amount of any such NIPs. Additional factors include the size of the patient, the age of the patient, the general condition of the patient, the particular disorder being treated, the severity of the disorder, the presence of other drugs in the patient, and the in vivo activity of the NIP, nNIF, nNIF analog, NRP, NRP analog, salt thereof, and the like. The trial dosages may be chosen after consideration of the results of animal studies and the clinical literature.
• Delivery methods and formulations useful for administering peptides to individuals are known in the art, and a skilled person would be able to determine the suitability of any particular method of delivery of a peptide to an individual for particular circumstances. For the purposes of illustration only, the following examples of methods and formulations for administering peptides to individuals are provided.
• Peptides may be administered to individuals orally; however, actions of the digestive system may reduce the bioavailability of the peptide.
• peptides may be administered in formulations containing enzyme inhibitors, or the peptides may be administered as part of a micelle, nanoparticle, or emulsion in order to protect the peptide from digestive activity.
• Peptides may also be administered by means of an injection.
• the peptides may be injected subcutaneously, intramuscularly, or intravenously. Further disclosure regarding methods of administering peptides through injection is found, e.g., in U.S. Pat. No. 5,952,301 .
• Peptides may further be administered by pulmonary delivery.
• a dry powder inhalation system may be used, wherein peptides are absorbed through the tissue of the lungs, allowing delivery without injection, while bypassing the potential reduction in bioavailability seen with oral administration. See Onoue et al., Expert Opin Ther Pat, 2008, 18: 429.
• the present disclosure provides in one of its aspects a package or kit, in the form of a sterile-filled vial or ampoule, that contains a NETosis and/or NET formation inhibiting amount of a NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof in either unit dose or multi-dose amounts, wherein the package or kit incorporates a label instructing use of its contents for the inhibition of such NETosis and/or NET formation.
• the package or kit contains the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof and the desired carrier, as an administration-ready formulation.
• the package or kit provides the NIP, nNIF, nNIF analog, NRP, NRP analog, and/or salt thereof in a form, such as a lyophilized form, suitable for reconstitution in a suitable carrier, such as phosphate-buffered saline.
• the package or kit is a sterile-filled vial or ampoule containing an injectable solution which comprises an effective, NETosis and/or NET formation inhibiting amount of NIP, nNIF, nNIF analog, NRP, NRP analog and/or salt thereof dissolved in an aqueous vehicle.
• Inflammatory pathways and immune mechanisms have checkpoints and modulatory brakes that prevent inappropriate initiation or unregulated propagation of effector events, which could otherwise cause pathologic collateral injury to the host (see Nathan C, Nature. 2002;420 (6917):846-52 and Medzhitov R., Nature. 2008;454 (7203):428-35). Tight control and modulation of inflammatory responses appear to be particularly important in the fetus and neonate, but the cellular and molecular mechanisms involved remain incompletely defined (see Dowling DJ, et al., Trends in immunology. 2014;35 (7):299310; Adkins B., et al., Immunologic research.
• nNIF in umbilical cord blood and A1ATm 358 in the placental matrix may represent regulatory factors that modulate NETosis in the perinatal milieu.
• Placental IL-8, a NETosis-inducing chemokine see Fuchs TA, et al., J Cell Biol.
• NET-inducing stimuli appear to be generated at the maternal -fetal interface, suggesting that unregulated NET formation can cause inflammatory pathology in the fetomaternal environment.
• Excessive intrapartum NET formation could also have additional negative consequences, including long-term neonatal immune dysregulation (see Brinkmann V, et al., J Cell Biol.
• nNIF expression may largely be a feature of the fetus and neonate, as are certain other immunoregulatory mechanisms (see Dowling DJ, et al., Trends in immunology. 2014;35 (7):299310; Adkins B., Immunologic research. 2013;57 (1 -3):246-57; and Elahi S, ef al., Nature. 2013;504 (7478): 158-62). [0159] Studies and previous observations suggest that nNIF and A1ATm are generated by proteolytic cleavage of A1AT in the placenta.
• A1AT is abundant in human placental tissue compartments (see Castellucci M, et al., Cell and tissue research. 1994;278 (2):283-9 and Frochaux V, et al., PloS one. 2014;9 (10):e109483). It has been proposed that progressive proteolytic cleavage of A1 AT occurs in the placenta, and proteases that mediate enzymatic fragmentation of A1AT to A1 ATm 358 in vitro are known (see Niemann MA, et al., Matrix. 1992; 12 (3):233-41 ; Niemann MA, et al., Biochim Biophys Acta.
• CRISPP was identified as a NRP.
• CRISPP-related peptides have been detected in plasma from patients with multiple types of cancer (see Cercek L, et al., Cancer Detect Prey. 1992; 16 (5-6): 305-19; Cercek L, ef al., Cancer Detect Prey. 1993; 17 (3):433-45; and Cercek L, et al., Cancer Detect Prey. 1993; 17 (3):447-54) but have not been linked to regulation of NETosis.
• NET formation facilitates experimental metastasis (see Cools-Lartigue J, et al., J Clin Invest. 2013; 123 (8):3446-58), and may also contribute to outcomes in cancer-associated infection and sepsis.
• endogenous CRISPP-related peptides may have significant influences on neoplastic complications by inhibiting formation of NETs.
• nNIF and CRISPP inhibit in vitro NET deployment induced by S. aureus, the bacterial toxin LPS, a previously-unrecognized viral trigger, dengue, a host-derived DAMP, heme, and the potent pharmacologic agonist, PMA.
• This analysis indicates that NRPs interrupt NET formation triggered by diverse stimuli (e.g., fungal agonists, etc.) that may be mediated by distinct activation pathways (see Sorensen OE, ef al., Journal of clinical investigation. 2016; 126 (5): 1612-20).
• nNIF and CRISPP inhibited in vivo NET formation in a murine model of E.
• FIGS. 8A-8F coli peritonitis
• NET formation is likely induced by the bacteria, LPS, and host-derived mediators, suggesting that NRPs can inhibit NET deployment by neutrophils stimulated by multiple agonists that act in combinational fashion in a complex inflammatory milieu and that, perhaps, induce NET formation via more than one pathway simultaneously (see Yipp BG, et al., Blood. 2013; 122 (16):2784-94).
• Validation of nNIF and CRISPP as inhibitors of NET deployment in this model also complements analysis of in vitro inhibition (see FIGS. 3A-3E) since it is suggested that pathways to NET formation vary in vivo and in vitro (see Sorensen OE, et a/., Journal of clinical investigation. 2016;126 (5):1612-20).
• nNIF and CRISPP were utilized as probes to explore the mechanism(s) by which NRPs inhibit NET formation. While NETosis induced by a number of agonists involves ROS generation (see Sorensen OE, et a/., Journal of clinical investigation. 2016;126 (5): 1612-20; Brinkmann V, et a/., J Cell Biol. 2012; 198 (5):773-83; Yipp BG, et al., Blood. 2013; 122 (16):2784-94; Brinkmann V, et ai, Science. 2004;303 (5663): 1532-5; Papayannopoulos V, et al., J Cell Biol.
• nNIF and CRISPP inhibit loss of lobules and expansion of nuclei in PMA-stimulated neutrophils (see FIGS. 7A, 12A, and 12B) in an assay based on earlier studies of chromatin decondensation in NETosis (see Papayannopoulos V, et al., J Cell Biol. 2010;191 (3):677-91 ).
• Neutrophil heterochromatin decondensation is mediated by PAD4, which catalyzes conversion of histone arginines to citrullines with consequent weakening of histone-DNA binding and unwinding of nucleosomes (see Sorensen OE, et al., Journal of clinical investigation. 2016; 126 (5):1612-20; Wang Y, er a/., J Cell Biol. 2009;184 (2):205-13; Li P, et al., J Exp Med. 2010;207 (9): 1853-62; and Kolaczkowska E, et al., Nature communications. 2015;6 (6673)).
• NRPs inhibit nuclear decondensation and NET formation at least in part by inhibiting PAD4 activity and nuclear histone deimination.
• NE is also thought to play a role in NETosis (see Sorensen OE, et al., Journal of clinical investigation. 2016; 126 (5): 1612-20).
• NE mediates nuclear histone cleavage in PMA-activated neutrophils (see Papayannopoulos V, et al., J Cell Biol. 2010; 191 (3):677-91 ); inhibitors of NE activity block nuclear decondensation (see Sorensen OE, et al., Journal of clinical investigation.
• NRPs do not directly inhibit NE activity in in vitro assays (see FIGS. 12A and 12B). NRPs, however, may interrupt NE-mediated events in NETosis pathways in other ways.
• a FLAG-tagged construct of CRISPP that inhibits NET formation was internalized by activated PMNs (see FIG. 7E) and was closely localized near NE in the neutrophil cytoplasm, suggesting, without being bound by a specific theory, that NRPs may have more than one site and mechanism of action.
• nNIF and CRISPP effectively inhibit NET formation by both human and murine neutrophils (see FIGS. 3A-3E and 8A-8F), whereas it has been reported that synthetic inhibitors of PAD4 have differential efficacy as inhibitors of NETosis by human and mouse neutrophils (see Lewis HD, et al., Nature chemical biology. 2015).
• nNIF and CRISPP were examined in mice challenged with LPS, which causes sterile systemic inflammation, NET formation, organ damage, and mortality (see Clark SR, et a/., Nature medicine. 2007; 13 (4):463-9; McDonald B, et a/., Cell host & microbe.
• NRPs provided an early survival advantage in this model (see FIG. 9A), suggesting, without being bound by any specific theory, that NETs are agents of inflammatory damage in the absence of pathogens and a consequent requirement for their containment and elimination. nNIF also improved mortality in the CLP model of polymicrobial sepsis (see FIGS.
• Example 1 - NET Formation by Human Neonatal Neutrophils Can Be Regulated by a Peptide in Umbilical Cord Blood
• nNIF or a larger protein that encompasses it, is an endogenous inhibitor of NET formation in cord blood plasma from preterm and term neonates (see FIG. 2B).
• Commercially available, active, full-length A1AT purified from human plasma and recombinant A1AT did not inhibit NET formation (see FIG. 2E), consistent with previous reports (see Farley K, ef a/., Journal of immunology. 2012; 189 (9):4574-81 and Frenzel E, et a/. , Int J Biol Sci. 2012;8 (7): 1023-5), indicating that intact A1AT does not contribute to NET-inhibitory activity.
• nN IF was detected in preterm cord blood plasma samples, whereas it was undetectable, or detectable in only trace levels, in plasma from healthy adults (see FIG. 2B, right panel).
• a peptide of appropriate molecular mass was not detected in plasma samples from adult subjects
• nNIF may be restricted to placental blood and blood of neonates in the first few days of life.
• Example 2 - nNIF and nN IF-Related Peptides (NRPs) are a Family of Previously Unrecognized PMN Modulators [0169] Additional NET inhibitory peptides were identified based on sequence analysis of nNIF. nNIF has substantial similarity to Cancer-Associated SCM- Recognition, Immunedefense Suppression, and Serine Protease Protection Peptide (CRISPP) (see FIG. 2A), a consensus peptide based on factors present in the blood of patients with cancer (see Cercek L, et al., Cancer Detect Prey. 1992; 16 (5-6):305-19; Cercek L, et al., Cancer Detect Prey.
• CRISPP Serine Protease Protection Peptide
• CRISPP-SCR a scrambled control peptide
• nNIF, CRISPP, and A1ATm 358 represent a previously-unrecognized family of nNIF-Related Peptides (NRPs) that modulate NET formation (see FIGS. 1A-5B).
• NRPs may be broadly distributed and that other NRPs may be identified.
• Phorbol Myristate Acetate is commonly employed as a potent non-physiologic agonist to induce NET formation in vitro (see Sorensen OE, et al., Journal of clinical investigation. 2016; 126 (5): 1612-20; Papayannopoulos V, et al., J Cell Biol.
• CRISPP-SCR blocked PMA-stimulated NET deployment (see FIG. 3B).
• CRISPP also inhibited NET formation induced by live Staphylococcus aureus (see Brinkmann
• NRPs inhibit NET deployment induced by microbes and microbial toxins, host-derived DAMPs, and pharmacologic agonists.
• Example 4 NRPs Selectively Inhibit NET Formation Without Interrupting Other Key Neutrophil Anti-Microbial Functions or Platelet Responses [0172] Total, phagocytic, and NET-mediated PMN killing of a pathogenic strain of
• Escherichia coli (E. coli) was examined using bacterial killing assays, and it was found that CRISPP depressed extracellular NET-mediated and total bacterial killing, but that phagocytic intracellular killing was not altered (see FIG. 6A).
• the NRPs did not inhibit generation of reactive oxygen species (ROS), phagocytosis, or interleukin 8 (IL-8) induced chemotaxis in Boyden chambers (see FIGS. 6B-6D). Although each peptide was not tested in all assays, these experiments indicate that the NRPs selectively inhibit
• ROS reactive oxygen species
• IL-8 interleukin 8
• FIGS. 6E and 6F indicate that PMNs, but not platelets, are cellular targets for NRPs and that platelet inflammatory activities are not disrupted by NRPs.
• nNIF and CRISPP were used as probes to dissect mechanisms of action for NRPs.
• Generation of ROS is thought to be essential in many, but not all, pathways that mediate NET formation (see Brinkmann V, et al. , J Cell Biol. 2012; 198 (5):773-83; Yipp BG, et al. , Blood. 2013; 122 (16):2784-94; Papayannopoulos V, et al. , J Cell Biol. 2010; 191 (3):677-91 ; Lood C, et al., Nature medicine. 2016;22 (2): 146-53; Yost CC, et al., Blood.
• nNIF CRISPP > A1 ATm 358 , which is the same as their relative inhibition of NETosis.
• nuclear histone H3 citrullination was examined in activated neutrophils (see Sorensen OE, et al., 2016;126 (5):1612-20 and Li P, et al., J Exp Med. 2010;207 (9): 1853-62), and rapid citrullination was detected within 15 minutes of activation with PMA. This was inhibited by nNIF and by Cl-amidine (see FIG. 7D), suggesting that NRPs act at this step to block nuclear decondensation (see FIG. 7A).
• Neutrophil elastase is also implicated in nuclear decondensation and NET formation (see Sorensen OE, et al., 2016; 126 (5): 1612-20; Brinkmann V, et al., J Cell Biol. 2012; 198 (5):773-83; Papayannopoulos V, et al., J Cell Biol. 2010; 191 (3):677-91 ; Kolaczkowska E, et al., Nature communications. 2015;6 (6673); and Branzk N, et al., Semin Immunopathol. 2013;35 (4):513-30).
• An NE inhibitor, sivelestat blocks nuclear decondensation in vitro (see FIGS.
• CRISPP and CRISPP-SCR were synthesized with FLAG tags added to the carboxy terminus of each peptide (CRISPP-F, CRISPP-SCR-F), and it was found that both are internalized by activated neutrophils (see FIG. 7E), that CRISPP-F inhibits NET formation (see FIG. 13), and, in a protein proximity assay, that CRISPP-F is initially localized within 40 nm of NE. This suggested that NRPs may block actions of NE in NETosis. In in vitro assays, however, neither nNIF nor CRISPP inhibited NE activity (see FIGS. 12A and 12B).
• NRPs inhibit NET formation in vivo and alter outcomes in systemic inflammation.
• a new model of in vivo NETosis was established using intraperitoneal (i.p.) infection of C57BL/6 mice with a clinical isolate of E. coli.
• live cell imaging of peritoneal fluid samples demonstrated robust NET formation.
• deposition of NETs was observed on the serosal surface of the peritoneal membranes.
• FIG. 8A Active peritonitis was demonstrated with increased neutrophil numbers and bacterial counts (see FIGS. 8C and 8D).
• the number of PMNs was greater in samples from CRISPP-treated animals, and the trend was to greater numbers in nNIF-treated mice (see FIG. 8C), potentially due to inhibition of lytic NETosis (see Brinkmann V, et al., J Cell Biol. 2012; 198 (5):773-83 and Yipp BG, et al., Blood. 2013;122 (16):2784-94).
• the number of E The number of E.
• coli colony forming units was also greater in samples from NRP-treated animals than in those treated with the CRISPP-SCR control (see FIG. 8D), suggesting decreased NET-mediated bacterial killing.
• nNIF and CRISPP also inhibited peritoneal NET formation in Swiss Webster mice infected with E. coli (see FIGS. 8E and 8F), suggesting that this result may be generalizable across mouse backgrounds.
• FIGS. 8A-9C demonstrate that nNIF and CRISPP inhibit NET formation in vivo, and provide initial evidence that they have beneficial effects in models of systemic sterile inflammation and infection in which NET formation may influence tissue injury and mortality (see Kolaczkowska E, et al., Nature communications. 2015;6 (6673); Clark SR, et al., Nature medicine. 2007; 13 (4):463-9; McDonald B, et al., Cell host & microbe. 2012;12 (3):32433; and Czaikoski PG, et al., PloS one. 2016;1 1 (2):e0148142).
• Example 6 An A1 AT Cleavage Fragment Generated by HTRA1 (HTRA1- CF) Inhibits NET Formation
• A1AT Progressive proteolytic cleavage of A1AT in the placenta may occur, and cleavage of A1AT by human stromelysin-3, yielding A1ATm 358 , has been reported (see Pei D, et al., J Biol Chem. 1994;269 (41 ):25849-55).
• other proteases can fragment A1AT.
• one or more placental proteases may cleave A1AT to yield nNIF. This could occur in extravascular placental compartments or in cord blood, depending on the protease(s) and local availability of substrate.
• HTRA1 high temperature requirement A1
• HTRA1 A protease, high temperature requirement A1 (HTRA1 ) is upregulated in the human placenta in the third trimester of pregnancy, and HTRA1 cleaves A1AT in the C-terminus, generating a fragment somewhat larger in size but including the sequence of nNIF (see Frochaux V ef a/., Plos One. 9(10): e109483. doi: 10.1371 ).
• HTRA1 -CF The peptide generated by cleavage of A1AT by HTRA1 (HTRA1 -CF) was synthesized, and it was found that HTRA1 -CF inhibits NET formation (see FIG. 14). Without being bound by any specific theory, as one or more placental proteases can enzymatically cleave A1AT, such cleavage may be a mechanism for production of nNIF.
• mice were housed in specific pathogen-free microisolator cages that were located in a room maintaining a constant temperature and on a 12-hour light-dark cycle. All treatment groups were weight matched and randomized to treatment at the initiation of an experiment. The researchers conducting the experiments were blinded to the experimental groups during testing. No inclusion or exclusion criteria were used in designing the experiments.
• Lipopolysaccharide E. coli serotype 01 1 1 : 134 and Salmonella enteritidis
• poly-L-lysine poly-L-lysine
• cytochalasin B poly-L-lysine
• cytochalasin D poly-L-lysine
• cytochalasin D paraformaldehyde
• p-FA paraformaldehyde
• sivelestat NE
• NE substrate MeOSuc
• pNA NE substrate
• thrombin thrombin
• Additional reagents were: TO-PRO ® -3 stain, phalloidin, SYTO ® Green (cell permeable DNA stain), and SYTOX ® Orange (cell impermeable DNA stain) (MOLECULAR PROBES ® ); Cl-amidine (CALBIOCHEM ® ); DNase (PROMEGATM); Anti-CD15-microbeads (MILTENYITM); Medium-199 (LONZATM), and micrococcal DNase (WORTH INGTON ® ).
• nNIF, NRPs, and their specific scrambled peptide controls were synthesized by the DNA Peptide Facility, a unit of the Health Sciences Center Cores at the University of Utah. The core facility also verified the sequence and purity of the provided peptides.
• PMNs were isolated from ACD or EDTA anticoagulated venous blood from healthy adults, healthy term infants, and prematurely born infants (see Yost CC, et a/., Blood. 2009;1 13 (25):6419-27 and Mclnturff AM, et a/., Blood. 2012; 120 (15):3118-25) under protocols approved by the University of Utah Institutional Review Board. For the eight prematurely born infants from whom cord and peripheral blood samples were collected, cord and peripheral blood plasma and PMN preparations were obtained at five separate time points throughout the first two months of life.
• PMN suspensions (>96% pure) were prepared by positive immunoselection using anti-CD15-coated microbeads and an AUTO-MACS ® cell sorter (MILTENYITM), and were resuspended at 2 ⁇ 10 6 cells/mL concentration in serum-free M-199 media at 370 °C in 5% CC>2/95% air. Human platelets were isolated as described (see Weyrich AS, et al., J Clin Invest. 1996;97 (6): 1525-34).
• PMNs were pre-incubated with autologous plasma, cord blood plasma, nNIF (0.2-70 nM), CRISPP (0.2-70 nM), nNIF-SCR (1 nM), or CRISPP-SCR (1 nM) for one hour prior to stimulation. After pre-incubation and/or stimulation, PMNs were gently washed with PBS and incubated with a mixture of cell permeable (SYTO ® Green, MOLECULAR PROBES ® ) and impermeable (SYTOX ® Orange, MOLECULAR PROBES ® ) DNA fluorescent dyes.
• Confocal microscopy was accomplished using a FV1000 1X81 confocal microscope and FLUOVIEWTM software (OLYMPUSTM). Both 20 ⁇ and 60* objectives were used. Z-series images were obtained at a step size of 1 ⁇ over a range of 20 ⁇ for each field. FLUOVIEWTM and ADOBETM PHOTOSHOPTM CS2 software was used for image processing.
• NET-associated histone H3 content was determined as previously described (see Mclnturff AM, et a/., Blood. 2012;120 (15):31 18-25). After live cell imaging of control and stimulated primary PMNs (2 10 6 /ml_; various agonists), the cells were incubated with PBS containing DNase (40 U/mL) for 15 minutes at room temperature to break down and release NETs formed in response to stimulation.
• SIGNALING ® SIGNALING ®
• LI-COR ® infrared-conjugated secondary antibodies
• Imaging and densitometry were performed on the ODYSSEY ® infrared imaging system (LI-COR ® ). This assay was previously validated as a surrogate for NET formation under in vitro conditions (see Mclnturff AM, et a/., Blood. 2012; 120
• Plasma samples were subjected to abundant plasma protein removal (NORGENTM).
• a polyclonal antibody raised against the carboxy terminal 18 amino acids of A1AT (LIFESPAN BIOSCIENCESTM) coupled to resin beads from an immunoprecipitation kit purchased from THERMO SCIENTIFICTM was then used to immunodeplete the samples.
• Non-immune IgG coupled to resin beads was used in parallel as a control.
• Plasma was diluted in lysis buffer from the kit and incubated with the anti-A1AT C-terminus antibody coupled beads or with control beads overnight at 4 °C. The beads were then separated by centrifugation, and the immunodepleted and control plasma samples were collected.
• the A1AT C-terminus antibody coupled and control beads were resuspended in kit-included elution buffer for 10 minutes at room temperature, followed by centrifugation and collection of the eluate and control supernatants.
• the eluate was analyzed by western blotting (16.5% Tris-tricine gel, BIO-RADTM) using the A1AT C-terminus antibody and by tandem mass spectroscopy.
• Immunodepleted plasma and eluate samples were examined in assays of NET formation. Active full-length native and recombinant A1AT (both from SIGMA-ALDRICH ® ) were suspended in elution buffer and tested in parallel.
• Chemotaxis by PMNs isolated from healthy adult donors was assessed using a modified Boyden chamber assay ⁇ a 1 hour pre-incubation with nNIF (1 nM), CRISPP (1 nM), or CRISPP-SCR (1 nM). Recombinant human IL-8 (2 ng/mL) was used as the chemoattractant. Chemotaxis through a 5 micron filter was determined by counting PMNs in 10 randomly selected high-power fields as previously described (see Hill HR, et a/., Lancet. 1974;2 (7881 ):617-9). In separate experiments, nNIF, CRISPP, or CRISPP-SCR (all at 1 nM) were evaluated for chemoattractant activity using the same system.
• PMNs were isolated from blood of healthy adult donors and resuspended in M-199 at a concentration of 2 ⁇ 10 6 cells/mL.
• Leukocytes were pre-incubated for 60 minutes under standard conditions with cytochalasin D and B (10 ⁇ ), nNIF (1 nM), CRISPP (1 nM), or CRISPP-SCR (1 nM).
• cytochalasin D and B 10 ⁇
• nNIF nNIF
• CRISPP CRISPP
• CRISPP-SCR CRISPP-SCR
• the PMNs were then washed and resuspended in the starting volume of M-199 before being spun down onto glass coverslips and fixed with 2% p-FA for 10 minutes and permeabilized with 0.1 % Triton-X-100 for 10 minutes.
• Leukocytes were stained with WGA 555 (INVITROGENTM) and TO-PRO ® -3 (MOLECULAR PROBES ® ), and randomly selected high-power visual field images were captured using confocal microscopy.
• IMAGEJTM software (NIH) was used to determine the percentage of PMNs that were positive for fluorescently labeled E. coli bioparticles detected at 488 nm.
• PMNs were isolated and resuspended to 2 10 6 cells/mL in M-199 media, pre-incubated with nNIF (1 nM), CRISPP (1 nM), nNIF-SCR (1 nM), CRISPP-SCR (1 nM), or the PAD4 inhibitor Cl-amidine (10 ⁇ ) for 1 hour at 37 °C in 5% C0 2 /95% air, and treated ⁇ PMA (20 nM) on poly-L-lysine coated glass coverslips for 2 hours. Nuclear decondensation was identified as described (see Papayannopoulos V, et a/., J Cell Biol. 2010; 191 (3):677-91 ).
• nNIF inhibition of PAD4 activity was determined using a PAD4 inhibitor screening assay kit (CAYMANTM). Briefly, nNIF (1 nM) was incubated with recombinant PAD4 and PAD4 enzyme substrate (2 mM) in PAD4 assay reagent for 30 minutes at 37 °C. The PAD4 inhibitor, Cl-amidine (10 ⁇ ), was used as a positive control for PAD4 inhibition. The reaction was stopped with PAD4 Stop Solution and detected using an included ammonia detector assay. Ammonia detector fluorescence was measured at 470 nm following excitation at 405 nm on a SPECTRAMAXTM M5 fluorescence plate reader (MOLECULAR DEVICESTM).
• FLAG-tagged CRISPP FLAG-tagged CRISPP
• FLAG-tagged CRISPP-SCR FLAG-tagged CRISPP-SCR peptides were synthesized by the University of Utah's core facility and detected using immunocytochemistry.
• Adult neutrophils were pre-incubated with either F-CRISPP (1 nM) or F-CRISPP-SCR (1 nM) for 1 hour at 37 °C in 5% C0 2 /95% air followed by stimulation with LPS (100 ng/mL) for 2 hours.
• the PMNs were then spun down onto glass coverslips with 2% p-FA fixation and 0.1 % Triton X-100 permeabilization.
• FLAG-tagged peptide was detected using a monoclonal anti-FLAG antibody (SIGMA-ALDRICH ® ) with TO-PRO ® -3 as a nuclear counterstain.
• SIGMA-ALDRICH ® monoclonal anti-FLAG antibody
• CRISPP CRISPP
• nNIF nNIF
• CRISPP-SCR CRISPP-SCR
• NETs in the peritoneal fluid were qualitatively and quantitatively analyzed using live cell imaging with confocal microscopy and NET-associated histone H3 release assays.
• NETs on the serosal surface of the peritoneal membrane were assessed quantitatively using live cell imaging, followed by standardized grid analysis of five randomly selected high-power visual fields per tissue sample (IMAGEJTM software, NIH).
• Peritoneal bacterial colony forming unit (cfu) counts were quantified by permeabilizing all recovered leukocytes with 0.1 % Triton X-100 for 10 minutes and performing serial dilutions and bacterial cultures on 5% sheep blood agar plates (HARDY DIAGNOSTICSTM). After a 24-hour incubation, bacterial counts were determined. Total leukocyte counts in the peritoneal lavage were determined in Neubauer chambers using an optical microscope after dilution in Turk's solution (2% acetic acid). Differential leukocyte analysis was performed using a 60 ⁇ oil immersion objective to assess morphology of cyto-centrifuged cells stained with May-Gruenwald-Giemsa dye. All mice were included in the final analysis.
• C57/BL6 mice were pretreated in blinded fashion with CRISPP (10 mg/kg), nNIF (10 mg/kg), or CRISPP-SCR (10 mg/kg) by i.p. injection 1 hour prior to and 6 hours after inoculation with LPS (25 mg/kg, i.p. injection).
• Control mice were i.p. injected with saline alone. Fluid resuscitation and antibiotic treatment were not used in these experiments. Survival was assessed over 50 or 72 hour intervals. All mice were included in the final survival analysis.
• C57BL/6 mice were anaesthetized with ketamine/xylazine (100 mg/kg and 10 mg/kg, i.p; respectively), and cecal ligation and puncture (CLP) was performed as previously described (see Araujo CV, et al., Shock. 2016;45 (4):393-403).
• nNIF (10 mg/kg) or nNIF-SCR (10 mg/kg) i.p. was given 1 hour prior to and 6 hours after CLP surgery.
• the animals received subcutaneous sterile isotonic saline (1 ml.) for fluid resuscitation immediately after the surgery. Sham-operated mice were subjected to identical procedures except that CLP was not done.
• Synthetic fluorogenic substrate of NE (MeOSuc)-AAPV-(pNA) (160 ⁇ ) was incubated with bioactive NE (500 nM) ⁇ the NE inhibitor, sivelestat (160 ⁇ ) or nNIF (50 nM), for 3 hours at 37 °C. The reactions were quenched with 5% glacial acetic acid and centrifuged at 14,000 rpm for 5 minutes.
• Chromatograms were obtained using an AGILENTTM 1 100 Series HPLC and a PHENOMENEX ® 5 pm C18 LUNA ® column (100 A, 4.6 150 mm) over a 30 minute 10% to 90% B gradient (Buffer A 0.1 % TFA in H 2 0, Buffer B 0.1 % TFA in ACN). Mass spectra were obtained for secondary validation of the reaction products using an API ® 3500 triple quadrupole mass spectrometer. Chromatograms were offset on both the X and Y axes (by 0.5 minutes and 0.1 A214, respectively) for greater visibility. Relative A214 was determined by normalizing all of the data to the tallest HPLC peak displayed in each graph.
• GRAPHPAD PRISMTM statistical software (version 5) was used to analyze results. The mean ⁇ standard error of the mean (SEM) was determined for each experimental variable. A Student t-test was used in FIGS. 2B, 6C, and 7B. ANOVA was used to identify differences that existed among multiple experimental groups. If significant differences were found, a Tukey's post hoc test (FIGS. 1A, 1 D, 2E, 3A- 3C, 3E, 6B-6F, 7C, 7D, 8A, 8E, and 8F), a Bonferroni's multiple comparison test (FIG. 6A), or the Newman-Keuls post-hoc procedure (FIGS. 8C and 9B) was used to determine the groups with significant differences.
• FIG. 8D A single tailed Mann-Whitney statistical tool was used for FIG. 8D.
• the Log-rank (Mantel- Cox) statistical tool was used to compare the survival curves between groups, and the post hoc Bonferroni correction was employed. All the data used in each statistical test met the assumption of the specific test and were normally distributed. All P values of ⁇ 0.05 were considered as statistically significant.
• Example 31 Placental HTRA1 Protease Cleaves Alpha-1 -antitrypsin and Generates Neonatal NET-lnhibitory Factor
• HTRA1 a serine protease
• HTRA1 and AAT plasma expression from term and preterm infants and adults were determined by ELISA.
• Bioactive (0.5 pg) or placental eluted HTRA1 was incubated with AAT (8 ⁇ ) for 18 hours at 37 °C. Carboxy-terminus fragments of AAT were detected using western blotting and mass spectrometry.
• reaction products were incubated for 1 hour with PMNs isolated from healthy adults prior to LPS stimulation (100 ng/mL) and assessed for NET formation using live cell imaging. Reactive oxygen species were assessed using flow cytometry, chemotaxis using a modified Boyden chamber assay, and bacterial killing using E. coli.
• HTRA1 Term and preterm infant placentas expressed HTRA1 , with significantly higher levels of HTRA1 in plasma from term (465.1 ⁇ 71.8 pg/mL) and preterm (385.9 ⁇ 71 .3 pg/mL) infant cord blood compared to adults (58.6 ⁇ 1 1 .6 pg/mL).
• Bioactive and placental-derived HTRA1 incubated with AAT generated a 4 kD AAT fragment.
• pre-incubation of this fragment with LPS-stimulated PMNs inhibited NET formation.
• the cleavage fragment from HTRA1 -AAT had no effect on reactive oxygen species generation, chemotaxis, or phagocytosis. However, incubation of this fragment with LPS-stimulated PMNs significantly reduced NET- associated bacterial killing compared to a scrambled HTRA1 -AAT fragment.
• HTRA1 expressed in the placenta interacts with AAT to generate a carboxy-terminus cleavage fragment with identical NET-inhibitory properties to nNIF. Accordingly, placental HTRA1 may generate nNIF in the fetal circulation as a mechanism of tolerance during gestation.

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  • 2017-09-05 BR BR112019004299A patent/BR112019004299A2/pt unknown
  • 2017-09-05 AU AU2017321965A patent/AU2017321965A1/en not_active Abandoned
  • 2017-09-05 JP JP2019512681A patent/JP2019534247A/ja active Pending
  • 2017-09-05 KR KR1020197009122A patent/KR20190045271A/ko not_active Application Discontinuation
  • 2017-09-05 CN CN201780067221.2A patent/CN109937047A/zh active Pending
  • 2017-09-05 US US16/328,697 patent/US10857201B2/en active Active
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• 2019
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